PROJECT SUMMARY Immune checkpoint blockade (ICB) has revolutionized the treatment of cancer but only about 20% of patients respond to treatment. The immune composition of the tumor microenvironment (TME) has been implicated in determining response to ICB. In particular, myeloid cells (MC) have been associated with both tumor progression and anti-tumor immunity. Among MC, classical type I dendritic cells (cDC1s) are uniquely capable of incorporating microenvironmental cues in the context of antigen (Ag) uptake and migrating to the lymph node (LN) where they present Ag and shape effector T cell responses. At the same time, cDC1s are also important in central and peripheral tolerance and the induction of T regulatory cells (Tregs). Using bulk and single cell RNA sequencing (scRNAseq) of mouse and human non-small cell lung cancer (NSCLC) lesions, we have identified a common transcriptional program in cDC1s that is upregulated upon migration to the LN. This migratory signature includes many immunoregulatory genes (e.g. PD-L1, CD200, ITGB8, SOCS2), therefore suggesting that tumors may hijack cDC1 tolerogenic programs to evade the immune system. Though these data provide a comprehensive transcriptional profile of migratory cDC1s, the function and relevance of many of the genes remain unknown. Our central hypothesis is that the upregulated genes in this signature control cDC1 homeostasis and phenotype. In Aim1, we will be using CRISPR/Cas9 Protein Barcode (Pro-code) technology to interrogate multiple KO to the migratory signature in steady state and in tumors, and performing high dimensional phenotyping in vivo to identify and characterize the genes that regulate the unique phenotype and molecular profile of cDC1s. In Aim 2, we will use in vitro OT-I/OT-II assays and tumor killing assays to determine how the migratory signature influences cDC1-T cell priming and activation. And in Aim 3, we will perform single KOs to the migratory cDC1 signature and determine changes to immune composition in the TME and tumor progression. The outcome of these studies will be a mechanistic, functional, and contextual understanding of the cDC1 transcriptional signature in the TME. This project may yield novel insights into DC biology and identify new ways to modulate or target this compartment for anti-tumor immunity.